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. 2024 Dec 13;10(50):eadr9364. doi: 10.1126/sciadv.adr9364

Fig. 5. NPEPPS enzymatic function regulates cisplatin response in vivo.

Fig. 5.

(A) Mice were injected with 4 × 106 cells in each flank to generate tumors using KU1919-Cis cells with NPEPPS−/−(WT-FLAG) or NPEPPS−/−(E353V-FLAG) expression. When engrafted tumors reached 100 mm3, mice were randomized to control or treatment groups and received either cisplatin (2 mg/kg; by intraperitoneal injection three times per week) or PBS control (equal volume of saline by intraperitoneal injection three times per week). Tumor size was measured with calipers every 2 to 3 days throughout the duration of treatment. Differences in growth rates by tumor genotype were evaluated by a two-way ANOVA mixed effects model. (B) Wet tumor weights were measured for each group. Comparisons were made using a one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (C) Fluorescence-based enzyme assay to quantify the catalytic activity of NPEPPS across the cells isolated from mouse tumors using H-Leu-AMC as the substrate. (D) DNA damage was measured by flow cytometry–based quantification of phosphorylated histone H2AX. Comparisons were made using a one-way ANOVA (*P < 0.05; ***P < 0.001). (E) Stratification of patient survival by above-median or below-median expression of the ratio of NPEPPS/Avg(LRRC8A-E) in BCa tumor samples before cisplatin treatment. Analysis of survival differences was conducted in R, and statistical significance was evaluated by Cox proportional hazard ratios and the log-rank test. Time to median survival is indicated by a dashed line. (F) Patient-derived organoids were evaluated for mRNA expression and cisplatin IC50. Left: Average counts per million mRNA expression of VRAC subunits LRRC8A-E and NPEPPS in each patient-derived organoid. Right: The correlation of NPEPPS/Avg(LRRC8A-E) expression with cisplatin sensitivity is plotted (r2 = 0.7172). Organoids from patient 1 were derived from a cystectomy sample, and the patient was not given neoadjuvant chemotherapy (NAC). Organoids from patients 2 to 5 were derived from the transurethral resection of bladder tumor samples before the patients received NAC.